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In this study, we aimed to assess the effect of four formalin-based fixation protocols (SFF, CFF, delayed formalin fixation-DFF, and cold formalin hyperfixation; CFH) on both DNA and RNA quality. We collected 97 colorectal cancer (CRC) and analyzed 23 metrics of nucleic acid quantity and quality yield using a multiplatform approach by combining spectrophotometric, fluorimetric, electrophoretic, and polymerase chain reaction (PCR) assays. Following confirmation of fixation-protocol-related different effects via clustering analysis, CFF presented best metrics compared with all protocols, specifically positive coefficients of DV1000-60000, DV2\/DV1, DNA \u03bb ratio 260\/230, and <jats:italic>ABL<\/jats:italic> gene expression absolute copies, and negative coefficient of DV150-1000. The SFF subgroup presented a positive coefficient of DV150-1000 and negative coefficients for DV1000-60000, DV2\/DV1, RNA \u03bb ratio 260\/230, RNA QuBit concentration, DV100\/200, RNA electrophoresis concentration and absolute quantity, and <jats:italic>ABL<\/jats:italic> copies. Overall, we confirmed the superior yield performances of CFF preservation for both DNA and RNA compared with the other protocols in our series of CRC samples. Pending further validations and clarification of the specific mechanisms behind these findings, our study supports the implementation of CFF in the pathology unit routine specimen management for tumor tissue molecular profiling.<\/jats:p>","DOI":"10.1007\/s00418-024-02326-5","type":"journal-article","created":{"date-parts":[[2024,9,24]],"date-time":"2024-09-24T19:02:04Z","timestamp":1727204524000},"page":"541-550","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Be bold, start cold! cold formalin fixation of colorectal cancer specimens granted superior DNA and RNA quality for downstream molecular analysis"],"prefix":"10.1007","volume":"162","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-1479-8121","authenticated-orcid":false,"given":"Ennio","family":"Nano","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7826-002X","authenticated-orcid":false,"given":"Alessandro","family":"Gambella","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4188-4247","authenticated-orcid":false,"given":"Michele","family":"Paudice","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-4464-2855","authenticated-orcid":false,"given":"Anna","family":"Garuti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5339-0629","authenticated-orcid":false,"given":"Simona","family":"Pigozzi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luca","family":"Valle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6477-3182","authenticated-orcid":false,"given":"Federica","family":"Grillo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0193-5281","authenticated-orcid":false,"given":"Luca","family":"Mastracci","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,9,24]]},"reference":[{"issue":"11","key":"2326_CR1","doi-asserted-by":"publisher","first-page":"1520","DOI":"10.5858\/arpa.2013-0691-RA","volume":"138","author":"BP Bass","year":"2014","unstructured":"Bass BP, Engel KB, Greytak SR, Moore HM (2014) A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? 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